1. Field of Invention
The invention relates to a novel nucleic acid molecule encoding xylose isomerase and the xylose isomerase encoded by the nucleic acid molecule. The invention relates to the research on enzymological properties of the xylose isomerase. The invention relates to the application of the xylose isomerase in the technical field of enzyme engineering. The invention also relates to a yeast strain containing the introduced nucleic acid sequence encoding the xylose isomerase.
2. Description of Related Art
Due to rising oil prices and global warming caused by greenhouse effect, people put more and more concern on the development and utilization of renewable energy sources. As an important renewable energy, biomass has a broad development space in the production of fuel ethanol as a raw material. For a long time, ethanol is produced by starch and sugars fermentation. Higher costs of the raw materials have greatly limited the development of the biomass fuel ethanol industry. Biomass produced by photosynthesis is up to 150 to 200 billion tons per year globally. Inside, above 80% of biomass are lignocellulosic substances, which are very cheap and easy to obtain. Therefore the production of the fuel ethanol by taking lignocelluloses as raw materials has an important practical significance. The lignocellulose mainly composes by cellulose, hemicelluloses, and lignin. It can be converted to pentose (xylose and arabinose) and hexose (glucose, galactose and mannose) by acidolysis or/and enzymolysis. The xylose is the second abandant monosaccharide, just follow the glucose, in the hydrolysate of lignocelluloses. The percentage of xylose could be up to 30%. Analysis shows that the ethanol yield could increases 25% if the xylose in lignocellulosic materials could be fully convered to ethanol (Nigam et al., J Appl Microbiol, 2001, 90 (2): 208-215). However, the Saccharomyce cerevisise, a good ethanol producer, cannot use the xylose as a carbon source. Therefore, constructing the S. cerevisise engineering strains that could efficiently utilize xylose to produce ethanol is one of the key issues in the sufficient utilization of lignocellulosic resources.
The natural xylose-utilizing yeasts convert xylose to xylulose, which S. cerevisise could metabolize, by two reactions. The xylose is firstly reduced to xylitol under the catalysis of the XR (xylose reductase), and then oxidized to xylulose by the XDH (xylitoldehydrogenase) (Träff et al., Appl Environ Microbiol, 2001, 67: 5668-5674). In most bacteria, such as Escherichia coli and Bacillus sp., the xylose is directly converted to the xylulose through xylose isomerase (XI) (Träff et al., Appl Environ Microbiol, 2001, 67: 5668-5674). Then, the xylulose is converted to xylulose 5-phosphate catalyzed by xylulose kinase (XK), and enters a pentose phosphate pathway (PPP). S. cerevisise has high ethanol productivity, and excellent characteristics such as high tolerance to inhibitors, unfavorable fermentation conditions, and high-concentration ethanol. Furthermore, it is a non-pathogenicity microorganism. However, due to the lack of XR and XDH, the S. cerevisis cannot metabolize xylose. To enable the S. cerevisiae obtain the ability to ferment the xylose, genes encoding NAD (P) H-depending XR and NAD+-depending XDH were cloned from Pichia stipitis and introduced into the S. cerevisiae to obtain the recombinant strain, which can be used for producing ethanol by fermenting the xylose (Walfridsson et al., Appl Environ Microbiol, 1995, 61: 4184-4190; Sedlak et al., Appl Biochem Biotechnol, 2004, 113-116: 403-416). But the affinity of XR to NADPH is much higher than that of XR to NADH, which results in cofactor imbalance in the process of oxidizing the xylitol into the xylulose under the catalysis of XDH. As a result, the by-product xylitol and glycerol accumulated and the yield of ethanol decreased. As the xylose isomerase does not need the cofactor, the xylose isomerase attracts the attention of researchers (Kuyper et al., FEMS Yeast Research, 2004, 4: 655-664).
Xylose isomerase, XI (EC5.3.1.5) can catalyze D-xylose into D-xylulose in vivo, and convert D-glucose into D-fructose in vitro, so that the xylose isomerase is also known as glucose isomerase. As the structure of the enzyme is very stable, the xylose isomerase is one of the good models for studying the relationships between protein structure and functions (Karimaki et al., Protein Eng Des Sel, 12004, 17 (12): 861-869). Moreover, the extremely important industrial application value makes the xylose isomerase is seen as important industrial enzyme as protease and amylase (Tian Shen et al., Microbiology Bulletin, 2007, 34 (2): 355-358; Bhosale et al., Microbiol Rev, 1996, 60 (2): 280-300). The scientists keep high concern and carried out extensive research on xylose isomerase. Since 1970s, the applications of the xylose isomerase have focused on the production of high fructose syrup and fuel ethanol. In recent years, scientists have found that under certain conditions, the xylose isomerase can be used for producing many important rare sugars, which are the production materials in the pharmaceutical industry, such as ribose, mannose, arabinose and lyxose (Karimaki et al., Protein Eng Des Se, 12004, 17 (12): 861-869). These findings bring new vitality in the research on the xylose isomerase.
It was reported that although a variety of bacterial sources xylose isomerase genes have been cloned and introduced into the S. cerevisiae, the mesophilic prokaryotic XIs cloned from common bacteria cannot functionally express in S. cerevisiae. In the about 450 currently published amino acid sequences of the xylose isomerase, two thermophilic bacteria sources XI expressed ˜1 U/mg specific activity products in the S. cerevisiae, which activity data were determined at 85 DEG C. However, only a very small part of the activity is retained at suitable temperature for S. cerevisiae growth (30 DEG C). Some other sources of xylose isomerase genes can express activity in the S. cerevisiae were found, including fungi sources, which were Piromyces sp-E2, Orpinomyces sp-Ukk1 and Cyllamyces aberensis; and bacteria sources, which were Clostridium phytofermentans, Clostridium difficile, Bacteroides Fusobacterium-mortiferum, and Ciona intestinalis. Wherein, only the xylose isomerase genes cloned from the Piromyces sp-E2 and Clostridium phytofermentans, especially the former, expressed elevated activity in S. cerevisiae. 